Liposoluble compounds useful as magnetic resonance imaging agents

ABSTRACT

Novel complexes of paramagnetic ions and compounds bearing long acyl chains have been synthesized as magnetic resonance imaging contrast agents. These novel liposoluble contrast agents may be administered alone, or with lipids, suspending agents or other additives. The lipids may be in the form of liposomes, micelles or lipid emulsions. The contrast agents of the invention have particular use in magnetic resonance imaging of the liver, blood pool and reticuloendothelial system.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 08/762,367,filed Dec. 9, 1996, now U.S. Pat. No. 5,762,910, which is a division ofU.S. application Ser. No. 08/487,287, filed Jun. 7, 1995, now U.S. Pat.No. 5,624,662, which is a division of U.S. application Ser. No.08/173,649, filed Dec. 27, 1993, now U.S. Pat. No. 5,466,438, which is adivision of U.S. application Ser. No. 07/887,290, filed May 22, 1992,now U.S. Pat. No. 5,312,617, which is a continuation-in-part of U.S.application Ser. No. 07/704,542, filed May 23, 1991, now abandoned. Thedisclosures of each of the foregoing applications are herebyincorporated herein by reference, in their entirety.

BACKGROUND OF THE INVENTION

Complexes of paramagnetic ions such as gadolinium-DTPA (Gd-DTPA) havebeen developed as magnetic resonance (MR) contrast agents. Whilegadolinium is quite toxic alone, the ion complex, Gd-DTPA, has much lesstoxicity, and has been used in MR imaging. Gd-DTPA, however, has limiteduse as an imaging agent. Indeed, while Gd-DTPA functions effectively asa contrast agent in the imaging of extracellular spaces, it provideslittle contrast enhancing effect as a blood pool imaging agent.Investigators have looked to other paramagnetic ions, such as manganese,for the development of similar complexes, such as Mn-DTPA. Suchcomplexes, however. have been largely unstable in the serum, and thussuffer limitations similar to Gd-DTPA. Recently manganese pyridoxalphosphate compounds have been developed as an MR contrast agent. Thesecompounds appear to function effectively as liver imaging agents, butare not thought to have much use as blood pool agents, or for otheruses, such as agents for imaging the bone marrow, spleen or lymph nodes.

Liposomes have also been studied as MR contrast agents. Liposomalparamagnetic contrast agents have been shown to be effective in imagingthe blood pool, liver, spleen and bone marrow. It has also been shownthat small liposomes under 50 nm in size were more effective as MRcontrast agents than larger liposomes, when the liposomes were used toentrap paramagnetic complexes such as Gd-DTPA. Even in the case of usingsmall liposomes, however, the entrapped Gd-DTPA has less relaxivity thanGd-DTPA which is free in solution and not entrapped within liposomes.Gd-DTPA entrapped within a lipid membrane has a reduction in relaxivitybecause of the reduction in water flux that occurs across theintervening lipid bilayer. To improve the relaxivity workers havedeveloped membrane bound paramagnetic ions but these have largely beenunstable and usually do not show improved relaxivity.

The need is great for new and/or better contrast agents for magneticresonance imaging. The present invention, which provides a new class ofliposoluble compounds having characteristics such as improved relaxivityand/or high stability, is directed to these important ends.

SUMMARY OF THE INVENTION

The present invention is directed to contrast agents useful in magneticresonance imaging.

Specifically, in one embodiment, the present invention pertains tocontrast agents for magnetic resonance imaging comprising a paramagneticion in combination with a compound of the formula ##STR1## wherein: eachR₁ is, independently, a substituted or unsubstituted C₇ -C₃₀ straightchain or cyclic compound;

each R₂ is, independently, a substituted or unsubstituted C₁ -C₃₀,straight chain or cyclic compound which may be internally interrupted byO, NH, NR₃, or S, where R₃ is a C₁ -C₃ alkyl; and

n is 0 to 1.

In another embodiment, the invention pertains to contrast agents formagnetic resonance imaging comprising a paramagnetic ion in combinationwith a compound of the formula ##STR2## wherein: each R₁ is,independently, a substituted or unsubstituted C₇ -C₃₀ straight chain orcyclic compound;

each R₂ is, independently, a substituted or unsubstituted C₁ -C₃₀straight chain or cyclic compound which may be internally interrupted byO, NH, NR₃, or S, where R₃ is a C₁ -C₃ alkyl; and

B is a substituted or unsubstituted C₁ -C₃₀ straight chain or cycliccompound which may be internally interrupted by O, NH, NR₃, or S.

Moreover, the subject invention encompasses contrast agents for magneticresonance imaging comprising a paramagnetic ion in combination with acompound of the formula ##STR3## wherein: each R₁ is, independently, asubstituted or unsubstituted C₇ -C₃₀ straight chain or cyclic compound;

each R₂ is, independently, a substituted or unsubstituted C₁ -C₃₀straight chain or cyclic compound which may be internally interrupted byO, NH, NR₃, or S, where R₃ is a C₁ -C₃ alkyl;

each m is 1 to 2; and

n is 1 to 20.

Further, the invention contemplates contrast agents for magneticresonance imaging comprising a paramagnetic ion in combination with acompound of the formula ##STR4## wherein: R₁ and R₂ are, independently,H, or a substituted or unsubstituted C₇ -C₃₀ straight chain or cycliccompound;

each R₃ and R₄ are, independently, H, or a substituted or unsubstitutedC₁ -C₃₀ straight chain or cyclic compound which may be internallyinterrupted by O, NH, NR₅, or S, where R₅ is a C₁ -C₃ alky; and

A is N, or a N-containing substituted or unsubstituted C₁ -C₃₀ straightchain or cyclic compound which may also be internally interrupted by O,NH, NR₅, or S, where R₅ is a C₁ -C₃ alkyl;

z is 1 to 10;

provided that at least one of R₁ and R₂ is other than H, and at leastone of R₃ and R₄ is other than H.

Still further, the invention provides a contrast agent for magneticresonance imaging comprising a paramagnetic ion in combination with acompound of the formula ##STR5## wherein: each R₁ is, independently, asubstituted or unsubstituted C₇ -C₃₀ straight chain or cyclic compound;

each R₂ is, independently, a substituted or unsubstituted C₁ -C₃₀straight chain or cyclic compound which may be internally interrupted byO, NH, NR₄, or S, where R₄ is a C₁ -C₃ alkyl;

R₃ is a substituted or unsubstituted C₁ -C₃₀ straight chain or cycliccompound which may be internally interrupted by O, NH, NR₄,or S, whereR₄ is a C₁ -C₃ alkyl; and

each m is, independently, 0 to 12.

Also encompassed in the subject invention are methods of providing animage of an internal region of a patient comprising (i) administering tothe patient one or more of the foregoing contrast agents, and (ii)scanning the patient using magnetic resonance imaging to obtain visibleimages of the region, and methods for diagnosing the presence ofdiseased tissue in a patient comprising (i) administering to the patientone or more of the foregoing contrast agents, and (ii) scanning thepatient using magnetic resonance imaging to obtain visible images of anydiseased tissue in the patient.

These and other aspects of the invention will become more apparent fromthe present specification and claims.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed, in part, to a new class of contrast agentswhich are highly useful in, for example, magnetic resonance imaging. Thenew class of agents, which comprise paramagnetic ions complexed withnovel acyl chain containing compounds, are described in more detailbelow.

Specifically, in one embodiment, the present invention pertains tocontrast agents for magnetic resonance imaging comprising a paramagneticion in combination with a compound of the formula ##STR6## wherein: eachR₁ is, independently, a substituted or unsubstituted C₇ -C₃₀ straightchain or cyclic compound;

each R₂ is, independently, a substituted or unsubstituted C₁ -C₃₀straight chain or cyclic compound which may be internally interrupted byO, NH, NR₃, or S, where R₃ is a C₁ -C₃ alkyl; and

n is 0 to 1.

In the above formula [I], R₁ may be a substituted or unsubstituted C₇-C₃₀ straight chain or cyclic compound. Preferably, R₁ is a C₇ -C₂₄ morepreferably a C₈ -C₁₈, straight chain or cyclic compound. By straightchain compound, as used herein, it is meant an open chain compound, as,for example, an aliphatic compound, such as an alkyl, alkenyl or alkynylcompound. Preferably the straight chain compound is an alkyl, such as,for example, decyl, dodecyl, hexadecyl or octadecyl. By cyclic compound,as used herein, it is meant a closed chain compound (as in a ring ofcarbon atoms), as, for example, a cyclic aliphatic or aromatic compound.Exemplary cyclic compounds include phenylene, and steroids such ascholesterol, estrogen or testosterone. By substituted or unsubstituted,as used herein, it is meant that the compound may have any one of avariety of substituents, in replacement, for example, of one or morehydrogen atoms in the compound, or may have no substituents. Exemplarysubstitutents include C₁ -C₅ alkyl and OH. Other suitable substituentswill be readily apparent to one skilled in the art, once armed with thepresent disclosure. Particularly preferred compounds are those: whereinR₁ is an unsubstituted C₇ -C₃₀ alkyl; wherein R₁ is an unsubstituted C₈-C₁₈ alkyl; wherein R₁ is decyl; wherein R₁ is dodecyl; and wherein R₁is octadecyl.

In formula [I], R₂ is, independently, a substituted or unsubstituted C₁-C₃₀ straight chain or cyclic compound which may be internallyinterrupted by O, NH, NR₃, or S, where R₃ is a C₁ -C₃ alkyl. Preferably,R₂ is a C₂ -C₁₂, more preferably a C₂ -C₆, straight chain or cycliccompound. Also preferably, the straight chain compound is an alkyl. Byinternally interrupted, as used herein, it is meant that the C₁ -C₃₀compound may have the carbon chain interrupted, as appropriate, withheteroatoms such as O, NH, NR₃, or S. If desired, the carbon chain mayhave no heteroatoms. By way of example, R₂ may comprise a polyhydricalcohol, such as --CH₂ --CHOH--CH₂ OH, --CH₂ --(CHOH)₂ --CH₂ OH, --CH₂--(CHOH)₃ --CH₂ OH, --CH₂ --(CHOH)₄ --CH₂ OH, or mannitol, sorbitol,glycidol, inositol, pentaerythritol, galacitol, adonitol, xylitol,alabitol. R₂ may also, for example, comprise a saccharide, includingmonosaccharides such as glucose, fructose, mannose, idose, galactose,allose, arabinose, gulose, fucose, erythrose, threose, ribose, xylose,lyxose, altrose, mannose, idose, talose, erythrulose, ribulose,xylulose, psicose, sorbose, tagatose, glucuronic acid, glucaric acid,galacturonic acid, manuronic acid, glucosamine, galactosamine andneuraminic acid, disaccharides such as sucrose, maltose, cellobiose,lactose, and trehalose, and polysaccharides such as a small starchmolecules, as well as homo or heteropolymers of the aforementionedsugars. Additionally, R₂ may comprise, for example, an ether such as--CH₂ (CHOH)nCH₂ OR₄, where R₄ is --(CH₂)m-CH₃, m is 0 to 26, X is O,--NH--, NR₃, or S, or R₂ may comprise a saccharide ether. R₂ may also,for example, comprise --{(CH₂)--(CH₂)m-X}--R₄, --(CH₂ CH₂ X)mR₄ or--(CHOH)m-OR₄. Particularly preferred compounds are those: wherein R₂ isa C₂ -C₆ alkyl; wherein R₂ is an uninterrupted C₂ -C₆ alkyl which issubstituted by OH; wherein R₂ is an unsubstituted C₂ -C₆ alkyl which isinternally interrupted by O.

Most preferred formula [I] compounds are those: wherein R₁ is octadecyl,R₂ is 2,3-dihydroxypropyl, and n is 0; wherein R₁ is decyl, R₂ is2,3-dihydroxypropyl, and n is 0; wherein R₁ is dodecyl, R₂ is2,3-dihydroxypropyl, and n is 0; wherein R₁ is octadecyl, R₂ is2,3-dihydroxypropyl, and n is 1.

In another embodiment, the invention is directed to a contrast agent formagnetic resonance imaging comprising a paramagnetic ion in combinationwith a compound of the formula ##STR7## wherein: each R₁ is,independently, a substituted or unsubstituted C₇ -C₃₀ straight chain orcyclic compound;

each R₂ is, independently, a substituted or unsubstituted C₁ -C₃₀straight chain or cyclic compound which may be internally interrupted byO, NH, NR₃, or S, where R₃ is a C₁ -C₃ alkyl; and

B is a substituted or unsubstituted C₁ -C₃₀ straight chain or cycliccompound which may be internally interrupted by O, NH, NR₃, or S.

In formula [II], R₁ and R₂ are as described in connection with theformula [I] compounds.

B is a substituted or unsubstituted C₁ -C₃₀ straight chain or cycliccompound which may be internally interrupted by O, NH, NR₃, or S, whereR₃ is a C₁ -C₃ alkyl. Particularly preferred compounds are those:wherein B is an unsubstituted and uninterrupted C₃ -C₃₀ cycloalkyl oraromatic; or wherein B is an unsubstituted and uninterrupted C₃ -C₆cycloalkyl or aromatic. By way of example, B may be cyclohexane,phenylene, or --CH₂ CH₂ X--(CH₂ CH₂ Y)n-CH₂ CH₂ --, where X and Y,independently, are O, --NH--, NR₃, or S.

A most preferred formula [II] compound is the compound: wherein R₁ isoctadecyl, R₂ is 2,3-dihydroxypropyl, and B is cyclohexyl.

The invention also contemplates a contrast agent for magnetic resonanceimaging comprising a paramagnetic ion in combination with apolyazacyclic compound of the formula ##STR8## wherein: each R₁ is,independently, a substituted or unsubstituted C₇ -C₃₀ straight chain orcyclic compound;

each R₂ is, independently, a substituted or unsubstituted C₁ -C₃₀straight chain or cyclic compound which may be internally interrupted byO, NH, NR₃, or S, where R₃ is a C₁ -C₃ alkyl;

each m is 1 to 2; and

n is 1 to 20.

In formula [III], R₁ and R₂ are as described in connection with theformula [I] compounds.

In formula [III], n is 1 to 20. Preferably, n is 1 to 10, morepreferably, 1 to 5, and most preferably 1 to 2.

Particularly preferred compounds are those: wherein R₁ is octadecyl, R₂is 2,3-dihydroxypropyl, m is 1, and n is 1.

Compounds that bear the polyazacyclic ring structure of formula [III]include 1,4,8,11-tetraazacyclotetradecane,1,4,7,10-tetraazacyclododecane, 1,4,7,10,13-pentaazacyclopentadecane.

Further, the invention contemplates contrast agents for magneticresonance imaging comprising a paramagnetic ion in combination with acompound of the formula ##STR9## wherein: R₁ and R₂ are, independently,H, or a substituted or unsubstituted C₇ -C₃₀ straight chain or cycliccompound;

each R₃ and R₄ are, independently, H, or a substituted or unsubstitutedC₁ -C₃₀ straight chain or cyclic compound which may be internallyinterrupted by O, NH, NR₅, or S, where R₅ is a C₁ -C₃ alkyl; and

A is N, or a N-containing substituted or and trehalose, unsubstituted C₁-C₃₀ straight chain or cyclic compound which may also be internallyinterrupted by O, NH, NR₅, or S, where R₅ is a C₁ -C₃ alkyl;

z is 1 to 10;

provided that at least one of R₁ and R₂ is other than H, and at leastone of R₃ and R₄ is other than H.

In the above formula [IV], R₁ and R₂ may be H, or a substituted orunsubstituted C₇ -C₃₀ straight chain or cyclic compound. Preferably, R₁and R₂ are a C₇ -C₂₄, more preferably a C₈ -C₁₈, straight chain orcyclic compound. Exemplary cyclic compounds include phenylene, andsteroids such as cholesterol, estrogen or testosterone. Preferably thestraight chain compound is an alkyl. Particularly preferred compoundsare those: wherein R₁ and R₂ are H, or an unsubstituted C₇ -C₃₀ alkyl;wherein R₁ and R₂ are H, or an unsubstituted C₈ -C₁₈ alkyl; and whereinR₁ and R₂ are H, or octadecyl.

In formula [IV], R₃ and R₄ are, independently, H, or a substituted orunsubstituted C₁ -C₃₀ straight chain or cyclic compound which may beinternally interrupted by O, NH, NR₅, or S, where R₅ is a C₁ -C₃ alkyl.Preferably, R₃ and R₄ are a C₂ -C₁₂, more preferably a C₂ -C₆, straightchain or cyclic compound. Also preferably, the straight chain compoundis an alkyl. By way of example, R₃ and R₄ may comprise a polyhydricalcohol, such as --CH₂ --CHOH--CH₂ OH, --CH₂ --(CHOH)₂ --CH₂ OH, --CH₂--(CHOH)₃ --CH₂ OH, --CH₂ --(CHOH)₄ --CH₂ OH, or mannitol, sorbitol,glycidol, inositol, pentaerythritol, galacitol, adonitol, xylitol,alabitol. R₃ and R₄ may also, for example, comprise a saccharide,including monosaccharides such as glucose, fructose, mannose, idose,galactose, allose, arabinose, gulose, fucose, erythrose, threose,ribose, xylose, lyxose, altrose, mannose, idose, talose, erythrulose,ribulose, xylulose, psicose, sorbose, tagatose, glucuronic acid,glucaric acid, galacturonic acid, manuronic acid, glucosamine,galactosamine and neuraminic acid, disaccharides such as sucrose,maltose, cellobiose, lactose, and trehalose, and polysaccharides such asa small starch molecules, as well as homo or heteropolymers of theaforementioned sugars. Additionally, R₃ and R₄ may comprise, forexample, an ether such as --CH2(CHOH)nCH2OR₆, where R₆ is --(CH₂)m-CH₃,m is 0 to 26, preferably 0 to 10, more preferably 0 to 5, X is O,--NH--, NR₅, or S, or R₃ and R₄ may comprise a saccharide ether. R₃ andR₄ may also, for example, comprise --{(CH₂)--(CH₂)m-X}--R₆, --(CH₂ CH₂X)mR₆, or --(CHOH)m-OR₆. Particularly preferred compounds are those:wherein R₃ and R₄ are H, or a C₂ -C₆ alkyl; wherein R₃ and R₄ are H, oran uninterrupted C₂ -C₆ alkyl which is substituted by OH; wherein R₃ andR₄ are H, or an unsubstituted C₂ -C₆ alkyl which is internallyinterrupted by O.

In formula [IV], z is 1 to 10. Preferably, z is 1 to 5, more preferably1 to 2.

A, in formula [IV] is N, or a N-containing substituted or unsubstitutedC₁ -C₃₀ straight chain or cyclic compound which may also be internallyinterrupted by O, NH, NR₅, or S, where R₅ is a C₁ -C₃ alkyl. Forexample, A may be N, or A may be R₇ --N--R₇, where each R₇ is,independently, --(CH₂ CH₂ X)n--, where n is 1 to 16, preferably 1 to 10,most preferably 1 to 2, and X is O, --NH--, NR₃, S or CHOH, where R₃ isa C₁ -C₃ alkyl. A may also be a N-containing cyclic compound such as apyrrole, pyrazole, imidazole, oxazole, thiazole, pyrroline, pyridine,pyrimidine, purine, quinoline, isoquinoline, or carbazole. Preferably, Ais N or a N-containing C₃ -C₃₀ cyclic compound. Most preferably, A is N.

A most preferred formula [IV] compound is that: wherein R₁ is octadecyl,R₂ is H, R₃ is methoxyethyl, R₄ is H, A is N, and z is 1.

In another aspect, the invention is directed to a contrast agent formagnetic resonance imaging comprising a paramagnetic ion in combinationwith a compound of the formula ##STR10## wherein: each R₁ is,independently, a substituted or unsubstituted C₇ -C₃₀ straight chain orcyclic compound;

each R₂ is, independently, a substituted or unsubstituted C₁ -C₃₀straight chain or cyclic compound which may be internally interrupted byO, NH, NR₄, or S, where R₄ is a C₁ -C₃ alkyl;

R₃ is a substituted or unsubstituted C₁ -C₃₀ straight chain or cycliccompound which may be internally interrupted by O, NH, NR₄, or S, whereR₄ is a C₁ -C₃ alkyl; and each m is, independently, 0 to 12.

In formula [V], R₁ and R₂ are as described in connection with theformula [I] compounds.

Also, in formula [V], R₃ is a substituted or unsubstituted C₁ -C₃₀straight chain or cyclic compound which may be internally interrupted byO, NH, NR₄, or S, where R₄ is a C₁ -C₃ alkyl. Preferably, R₃ is a C₂-C₁₂, more preferably a C₂ -C₆, straight chain or cyclic compound. Alsopreferably, the straight chain compound is an alkyl or alkenyl. By wayof example, R₃ may be ethylene, propylene, butylene, etc. Also by way ofexample, R₃ may comprise a polyhydric alcohol, such as --CH₂ --CHOH--CH₂OH, --CH₂ --(CHOH)₂ --CH₂ OH, --CH₂ --(CHOH)₃ --CH₂ OH, --CH₂ --(CHOH)₄--CH₂ OH, or mannitol, sorbitol, glycidol, inositol, pentaerythritol,galacitol, adonitol, xylitol, alabitol. R₃ may also, for example,comprise a saccharide, including monosaccharides such as glucose,fructose, mannose, idose, galactose, allose, arabinose, gulose, fucose,erythrose, threose, ribose, xylose, lyxose, altrose, mannose, idose,talose, erythrulose, ribulose, xylulose, psicose, sorbose, tagatose,glucuronic acid, glucaric acid, galacturonic acid, manuronic acid,glucosamine, galactosamine and neuraminic acid, disaccharides such assucrose, maltose, cellobiose, lactose, and trehalose, andpolysaccharides such as a small starch molecules, as well as homo orheteropolymers of the aforementioned sugars. Additionally, R₃ maycomprise, for example, an ether such as --CH₂ (CHOH)nCH₂ OR₅, where R₅is --(CH₂)n-CH₃, n is 0 to 26, X is O, --NH--, NR₄, or S, or R₃ maycomprise a saccharide ether. R₃ may also, for example, comprise--{(CH₂)--(CH₂)n-X}--R₅, --(CH₂ CH₂ X)nR₅ or --(CHOH)n-OR₅. Otherexemplary cyclic compounds include phenylene, and steroids such ascholesterol, estrogen or testosterone. Exemplary substitutents includeC₁ -C₅ alkyl and OH. Other suitable substituents will be readilyapparent to one skilled in the art, once armed with the presentdisclosure. Particularly preferred formula [V] compounds are those:wherein R₃ is an unsubstituted C₂ -C₁₂ alkyl or alkenyl; wherein R₃ isan unsubstituted C₂ -C₆ alkyl or alkenyl; and wherein R₃ is ethylene.Other particularly preferred compounds are those: wherein R₃ is anuninterrupted C₂ -C₆ alkyl or alkenyl which is substituted by OH;wherein R₃ is an unsubstituted C₂ -C₆ alkyl or alkenyl which isinternally interrupted by O.

In formula [V], m is 1 to 12. Preferably, m is 1 to 10, more preferably,1 to 5, and most preferably 1 to 2.

A particularly preferred formula [V] compound is that: wherein R₁ isoctadecyl, R₂ is 2,3-dihydroxypropyl, R₃ is ethylene, and m is 0.

The formula [V] compounds are extremely well suited to the chelation ofmultiple paramagnetic ions, including different types of ions.

As the above indicates, the length of the acyl chains covalently boundto the formula [I], [II], [III], [IV] and [V] compounds be varied up to30 carbon atoms in length. Longer length chains, e.g. 18 carbon atoms,are preferred for use of the contrast agent with lipid compounds.Shorter carbon chains, e.g. 8 carbon atoms, are preferred when preparingthe agents for use either alone or with suspending agents, generallybecause of their somewhat greater water solubility. Also, two acylchains attached to the complex are preferred.

The liposoluble compounds of formulas [I], [II], [III], [IV] and [V] maybe employed singlely or in combination with one another, and incombination with one or more paramagnetic ions as contrast agents formagnetic resonance imaging. Exemplary paramagnetic ions includetransition, lanthanide (rare earth) and actinide ions, as will bereadily apparent to those skilled in the art, in view of the presentdisclosure. Preferable paramagnetic ions include those selected from thegroup consisting of Cr⁺³, Co⁺², Mn⁺², Ni⁺², Fe⁺³, Fe⁺², La⁺³, Cu⁺²,Gd⁺³, Ce⁺³, Tb⁺³, Pr⁺³, Dy⁺³, Nd⁺³, Ho⁺³, Pm⁺³, Er⁺³, Sm⁺³, Tm⁺³, Eu⁺³,Yb⁺³ and Lu⁺³. More preferably, the paramagnetic ion is selected fromthe group consisting of Mn⁺², Fe⁺³ and Gd⁺³, most preferably Mn⁺². Ifdesired, two or more different ions may be used in combination. As thoseskilled in the art will recognize, once armed with the presentdisclosure, various combinations of the lipsoluble compounds andparamagnetic ions may be used to modify the relaxation behavior of theresulting contrast agent. The subject paramagnetic ion and liposolublecompound complexes of the invention have been found to be extremelyeffective contrast enhancement agents for magnetic resonance imaging.

The contrast agents of the invention may further comprise a lipidcompound. Such lipid compounds may include any one of a variety of classor type of lipids, such as, for example, cholesterols,phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines,phosphatidylglycerols, phosphatidic acids, phosphatidylinositols,phospholipids, lysolipids, fatty acids, sphingomyelin,glycosphingolipids, glucolipids, glycolipids, sulphatides, lipids withether and ester-linked fatty acids and polymerizable lipids, andcombinations thereof. The phospholipids are one generally preferred typeof lipid, and include phospholipids, phosphatidylcholines,phosphatidylethanolamines, phosphatidylserines, phosphatidylglycerols,phosphatidic acids, phosphatidylinositols, diacetyl phosphates. Onepreferred type of phospholipid is a phosphatidyl choline lipid compound,such as egg phosphatidylcholine, dipalmitoyl phosphalidycholine,monomyristoyl phosphatidylcholine, monopalmitoyl phosphatidylcholine,monostearoyl phosphatidylcholine, monooleoyl phosphatidylcholine,dibutroyl phosphatidylcholine, divaleroyl phosphatidylcholine, dicaproylphosphatidylcholine, diheptanoyl phosphatidylcholine, dicapryloylphosphatidylcholine, distearoyl phosphatidylcholine, or otherphosphatidyl compounds such as phosphatidylserine, phosphatidylinositol,and diphosphatidylglycerol. Another preferred lipid is a fatty acidlipid compound, such as linoleic acid, oleic acid, palmitic acid,linolenic acid, stearic acid, lauric acid, myristic acid, arachidicacid, palmitoleic acid, arachidonic acid ricinoleic acid,tuberculosteric acid, lactobacillic acid. A still other preferred lipidis a glycolipid compound such as cerebrosides, gangliosides (such asmonosialoganglioside and GM1), and ceramides (such as lactosylceramide).A further preferred lipid is a ceramide which is ceramides

As those skilled in the art will recognize, once placed in possession ofthe present invention, the lipids employed in the invention may beselected to optimize the particular diagnostic use, minimize toxicityand maximize shelf-life of the product. For example, neutral vesiclescomposed of phosphatidylcholine and cholesterol function quite well asintravascular contrast agents. To improve uptake by cells such as thereticuloendothelial system (RES), a negatively charged lipid such asphosphatidylglycerol, phosphatidylserine or similar material may beadded. To prolong the blood pool half-life, highly saturated lipids thatare in the gel state at physiological temperature such as dipalmitoylphosphatidylcholine may be used. For even greater vesicle stability andprolongation of blood pool half-life the lipid can be polymerized usingpolymerizable lipids, or be coated with polymers such as polyethyleneglycol so as to protect the lipid from serum proteins. In addition,gangliosides such as GM1 can be incorporated in the lipid.

The lipid compound employed in connection with the present invention maybe in the form of a lipid emulsion, liposome, or micelle, orcombinations thereof. Lipid emulsions, liposomes, and micelles, andmethods for their preparation, are well known in the art.

For example, liposomes, that is, lipid vesicles comprising aqueouscompartments enclosed by a lipid bilayer, may be prepared using any oneof a variety of conventional liposome preparatory techniques which willbe apparent to those skilled in the art. These techniques includefreeze-thaw, as well as techniques such as sonication, chelate dialysis,homogenization, solvent infusion, micro-emulsification, spontaneousformation, solvent vaporization, reverse phase, French pressure celltechnique, controlled detergent dialysis, and others, each involvingpreparing the liposomes in various fashions. Preparation may be carriedout in a solution, such as a phosphate buffer solution, containingliposoluble contrast agents of the invention, so that the contrast agentis incorporated in to the liposome membrane. Alternatively, the contrastagents may be added to already formed liposomes. The size of theliposomes can be adjusted, if desired, by a variety of proceduresincluding extrusion, filtration, sonication, homogenization, employing alaminar stream of a core of liquid introduced into an immiscible sheathof liquid, and similar methods, in order to modulate resultant liposomalbiodistribution and clearance. Extrusion under pressure through pores ofdefined size is, however, the preferred means of adjusting the size ofthe liposomes. Although liposomes employed in the subject invention maybe of any one of a variety of sizes, preferably the liposomes are small,that is, less than about 100 nm in outside diameter, more preferablyless than about 50 nm. The foregoing techniques, as well as others, arediscussed, for example, in U.S. Pat. No. 4,728,578; U.K. PatentApplication GB 2193095 A; U.S. Pat. No. 4,728,575; U.S. Pat. No.4,737,323; International Application PCT/US85/01161; Mayer et al.,Biochimica et Biophysica Acta, Vol. 858, pp. 161-168 (1986); Hope etal., Biochimica et Biophysica Acta, Vol. 812, pp. 55-65 (1985); U.S.Pat. No. 4,533,254; Mayhew et al., Methods in Enzymology, Vol. 149, pp.64-77 (1987); Mayhew et al., Biochimica et Biophysica Acta, Vol 755, pp.169-74 (1984); Cheng et al, Investigative Radiology, Vol. 22, pp. 47-55(1987); PCT/US89/05040, U.S. Pat. No. 4,162,282; U.S. Pat. No.4,310,505; U.S. Pat. No. 4,921,706; and Liposome Technology,Gregoriadis, G., ed., Vol. I, pp. 29-31, 51-67 and 79-108 (CRC PressInc., Boca Raton, Fla. 1984). The disclosures of each of the foregoingpatents, publications and patent applications are incorporated byreference herein, in their entirety. Although any of a number of varyingtechniques can be employed, preferably the liposomes employed in theinvention are prepared via microemulsification techniques, using, forexample, a microfluidizer (Microfluidics, Newton, Mass.).

Micelles, that is, clusters or aggregates of lipid compounds, generallyin the form of a lipid monolayer, may be prepared using any one of avariety of conventional liposome preparatory techniques which will beapparent to those skilled in the art. These techniques typically includethe steps of suspension in an organic solvent, evaporation of thesolvent, resuspension in an aqueous medium, sonication and thencentrifugation. The foregoing techniques, as well as others, arediscussed, for example, in Canfield et al., Methods in Enzymology, Vol.189, pp. 418-422 (1990); El-Gorab et al, Biochem. Biophys. Acta, Vol.306, pp. 58-66 (1973); Colloidal Surfactant, Shinoda, K., Nakagana,Tamamushi and Isejura, Academic Press, N.Y. (1963) (especially "TheFormation of Micelles", Shinoda, Chapter 1, pp. 1-88); Catalysis inMicellar and Macromolecular Systems, Fendler and Fendler, AcademicPress, N.Y. (1975). The disclosures of each of the foregoingpublications are incorporated by reference herein, in their entirety.Themicelles may be prepared in the presence of liposoluble contrast agentsof the invention, or the contrast agent may be added to already formedmicelles. Preferable lipid compounds used in preparing the micellesinclude, for example, monomyristoyl phosphatidylcholine, monopalmitoylphosphatidylcholine, monostearoyl phosphatidylcholine, monooleoylphosphatidylcholine, dibutroyl phosphatidylcholine, divaleroylphosphatidylcholine, dicaproyl phosphatidylcholine, diheptanoylphosphatidylcholine, dicapryloyl phosphatidylcholine. Other preferablelipid compounds for the micelles of the invention include, for example,linoleic acid, oleic acid, palmitic acid, linotenic acid, stearic acid,phosphatidylcholine, and phosphatidylethanolamine.

Lipid emulsions are also well known and may be prepared usingconventional techniques. As those skilled in the art will recognize, alipid emulsion is a substantially permanent heterogenous liquid mixtureof two or more liquids that do not normally dissolve in each other, bymechanical agitation or by small amounts of additional substances knownas emulsifiers. Typically, in preparing the emulsion, the lipids areadded to ethanol or chloroform or any other suitable organic solvent andagitated by hand or mechanical techniques. The solvent is thenevaporated from the mixture leaving a dried glaze of lipid. The lipidsare resuspended in aqueous media, such as phosphate buffered saline,resulting in an emulsion. To achieve a more homogeneous sizedistribution of the emulsified lipids, the mixture may be sonicatedusing conventional sonication techniques, further emulsified usingmicrofluidization (using, for example, a Microfluidizer, Newton, Mass.),and/or extruded under high pressure (such as, for example, 600 psi)using an Extruder Device (Lipex Biomembranes, Vancouver, Canada).Contrast agents of the invention may be added to the lipids duringpreparation of the emulsion, such as at the stage where the lipids areadded to the organic solvent or at other stages of preparation, or maybe added after the lipid emulsion has been formed, as desired. Inpreparing the lipid emulsions, particularly useful additives are, forexample, soybean lecithin, glucose, Pluronic F-68, and D,L-α-tocopherol(Vitamin E), generally in an amount of about 0.03 to about 5 percent byweight. These additives are particularly useful where intravenousapplications are desired. Techniques and ingredients for formulatinglipid emulsions are well known in the art. Suitable procedures andemulsion ingredients are reported, for example, in Modern Pharmaceutics,pp. 505-507, Gilbert Baker and Christopher Rhodes, eds., Marcel DekkerInc., New York, N.Y. (1990), the disclosures of which are herebyincorporated herein by reference in their entirety.

As those skilled in the art will recognize, any of the lipid compoundsand preparations containing the lipid compounds (including the lipid andcontrast agent preparations), may be lyophilized for storage, andreconstituted in, for example, an aqueous medium (such as sterile wateror phosphate buffered saline), with the aid of vigorous agitation. Inorder to prevent agglutination or fusion of the lipids as a result oflyophilization, it may be useful to include additives in the formulationto prevent such fusion or agglutination. Additives which may be usefulinclude sorbitol, mannitol, sodium chloride, glucose, trehalose,polyvinylpyrrolidone and polyethyleneglycol (such as PEG 400). These andother additives are described in the literature, such as in the U.S.Pharmacopeia, USP XXII, NF XVII, The United States Pharmacopeia, TheNational Formulary, United States Pharmacopeial Convention Inc., 12601Twinbrook Parkway, Rockville, Md. 20852, the disclosures of which arehereby incorporated herein by reference in their entirety. Lyophilizedpreparations generally have the advantage of greater shelf life.

The contrast agent of the invention may further, if desired, comprise asuspending agent. Preferable suspending agents include polyethyleneglycol, lactose, mannitol, sorbitol, ethyl alcohol, glycerin, lecithin,polyoxyethylene sorbitan monoleate, sorbitan monoleate and albumin. Asthose skilled in the art would recognize, various sugars and otherpolymers may also be employed, such as polyethylene,polyvinylpyrrolidone, propylene glycol, and polyoxyethylene. The amountof paramagnetic acylated MR contrast agent, e.g., Mn-DDP-EDTA, may varyfrom about 1 to 75 percent by weight of the total ingredients used toformulate the paramagnetic MR contrast agent emulsion.

The present invention is useful in imaging a patient generally, and/orin specifically diagnosing the presence of diseased tissue in a patient.The imaging process of the present invention may be carried out byadministering a contrast medium of the invention to a patient, and thenscanning the patient using magnetic resonance imaging to obtain visibleimages of an internal region of a patient and/or of any diseased tissuein that region. By region of a patient, it is meant the whole patient,or a particular area or portion of the patient. The contrast medium isparticularly useful in providing images of the blood pool, liver,reticuloendothelial system, spleen, bone marrow, lymph nodes, andmuscle. It is especially useful in imaging the blood pool, liver,reticuloendothelial system, spleen, particularly the blood pool. Becauseof their high relativity, these contrast agents are especially effectiveblood pool agents. Also, as shown by their in vivo effectiveness at lowdoses, these agents are highly effective at enhancing the liver andhighly useful for improving the detection of hepatic metastases. Thepatient can be any type of animal, but preferably is a mammal, and mostpreferably a human.

Any of the various types of magnetic resonance imaging devices can beemployed in the practice of the invention, the particular type or modelof the device not being critical to the method of the invention. Themagnetic resonance imaging techniques whch are employed are conventionaland are described, for example, in Kean, D. M., and M. A. Smith,Magnetic Resonance Imaging: Principles and Applications (Williams andWilkins, Baltimore 1986), the disclosures of which are herebyincorporated herein by reference in their entirety. Contemplatedmagnetic resonance imaging techniques include, but are not limited to,nuclear magnetic resonance (NMR), NMR spectroscopy, and electronic spinresonance (ESR). The preferred imaging modality is NMR.

As one skilled in the art would recognize, administration of thecontrast agent to the patient may be carried out in various fashions,such as intravascularly, orally, rectally, etc., using a variety ofdosage forms. Preferably, administration is by intravascularly. Theuseful dosage to be administered and the particular mode ofadministration will vary depending upon the age, weight and theparticular animal and region thereof to be scanned, and the particularcontrast agent of the invention to be employed. Typically, dosage isinitiated at lower levels and increased until the desired contrastenhancement is achieved. By way of general guidance, typically betweenabout 0.1 mg and about 1 g of the liposoluble compound of formulas [I],[II], [III], [IV], and [V], and between about 1 and about 50 micromolesof paramagnetic ion, each per kilogram of patient body weight, isadministered, although higher and lower amounts can be employed.Similarly, by way of general guidance, where lipids or suspending agentsare used in the formulation, generally between about 0.5 and about 50percent by weight of the entire formulation of each may be employed,although higher and lower amounts may also be used.

In carrying out the method of the present invention, the contrast agentmay be used alone, or in combination with other diagnostic, therapeuticor other agents. Such other agents include excipients such as flavoringor coloring materials.

In employing the contrast agents, they are preferably suspended inaqueous solution and the contrast medium formulated using steriletechniques. An advantage to using smaller liposomes (e.g., 100 nm andbelow in size) and micelles or emulsified lipids, as well as the simplesuspension of paramagnetic ions and liposoluble compounds, is that thecontrast agents may be filtered through 0.22 micron line filters eitherimmediately prior to administration, such as by intravenous injection,or as a terminal step in formulation of the contrast agents, to removeany potential pyrogens.

For formulating these contrast agents into stable preparations otheradditives may be employed. For example, in formulating contrast agentsfor intravenous injection, parenteral additives may be included in thepreparation. Such additives to include tonicity adjusting additives suchas dextrose and sodium chloride, to formulate an isosmotic contrastmedium. These tonicity additives are generally provided in minoramounts, such as about 0.1% to about 0.5% by weight of the totalformulation. In addition, antimicrobial additives may be included in thefinal preparation so as to avoid bacterial growth. Such antimicrobialadditives, in generally acceptable amounts, may include but are notlimited to benzalkonium chloride (typically 0.01% by weight of the totalformulation), benzyl alcohol (typically 1-2% by weight), chlorobutanol(typically 0.25-0.5% by weight), metacresol (typically 0.1-0.3% byweight), butyl p-hydroxybenzoate (typically 0.015% by weight), methylp-hydroxybenzoate (typically 0.1-0.2% by weight), propylp-hydroxybenzoate (typically 0.2% by weight), phenol (0.25-0.5% byweight) and thimerosal (typically 0.01% by weight). Additionally,antioxidants may be included in the preparation, and are particularlyuseful where the contrast agent contains unsaturated lipids. Suchantioxidants in their generally useful amounts include ascorbic acid(typically 0.01-0.5% by weight), cysteine (typically 0.1-0.5% byweight), monothioglycerol (typically 0.1-1.0% by weight), sodiumbisulfite (typically 0.1-1.0% by weight), sodium metabisulfite(typically 0.1-1.0% by weight), and tocopherols (typically 0.05-0.5% byweight). As those skilled in the art will recognize, the contrast agentsof the invention may be formulated in a variety of means to beparticularly suitable for intravascular delivery, delivery into any bodycavity, or other delivery targets.

The contrast agents of the invention exhibit both high T1 and T2relaxivity, especially high where lipids are also employed. Although notintending to be bound by any theory of operation, where lipid compoundsare employed along with the liposoluble compounds and paramagnetic ions,it is believed that the high relaxivity of the subject contrast agentsmay be due to the liposoluble nature of the compounds, and, in part, theconcomitant ability of those compounds to fix the contrast agent in themembranes of those lipid compounds. This, in turn, may serve tocritically limit the tumbling of the contrast agents, thereby increasingrelaxivity.

Another advantage of the present contrast agents are their stability.Indeed, not only does the increased stability result in a higher shelflife, but, more importantly, the stability of these compounds results indecreased toxicity. Unlike many of the contrast agent chelates in theprior art, the subject compounds are highly stable, even in mediacontaining serum. As the examples show, the testing of stability inserum indicates that almost no metal ion dissociated from these novelcontrast agents.

The present invention is further described in the following examples. Inthese example, Examples 1-8 and 10-17 are actual examples. Example 9 isa prophetic example. These examples are for illustrative purposes only,and are not to be construed as limiting the appended claims.

EXAMPLE 1

Synthesis of ManganeseN,N'-Bis-(Carboxy-Octadecylamido-Methyl-N-2,3-Dihydroxypropyl)-Ethylenediamine-N,N'-Diacetate(Mn-EDTA-ODP) (Formula I, wherein R₁ is octadecyl, R₂ is2,3-dihydroxypropyl, and n is 0)

Structure ##STR11## Synthetic Route (i) Synthesis of3-Octadecylamino-1,2-Dihydroxy-Propane (ODP)

Octadecylamine (18 g) was dissolved in 200 ml methanol and heated to 60°C. Glycidol (4.95 g) was added dropwise under constant stirring over oneand half hours. The reaction mixture was kept under reflux for oneadditional hour, and then cooled to room temperature, and evaporated todryness, resulting in 22 g white solid material. This was thenrecrystallized from hexane, to yield ODP, mp 81-83° C.

(ii) Synthesis ofN,N'-Bis(Carboxy-Octadecylamidomethylene-N-1,2,-Dihydroxypropyl)-EthylenediamineN,N'-Diacetic Acid (EDTA-ODP)

EDTA anhydride (1.28 g) and 3-octadecylamino-1,2,-dihydroxypropane (3.43g) were dissolved together in fresh dried methanol (160 ml). Thereaction mixture was stirred at 35-40° C. for 12 hours, while the EDTAanhydride particles disappeared and the solution became transparent. Thereaction mixture was then rotary evaporated to dryness and 4.6 g whitesolid was obtained, yielding EDTA-ODP, m.p. (decomposition) 130° C.

Elemental Analysis: C₅₂ H₁₀₂ N₄ O₁₀ ; Calc. C 66.20; H 10.90; N; 5.94;Anal. C 67.15; H 11.46; N; 5.90;

(iii) Synthesis ofManganese-N,N'-Bis(carboxy-Octadecylamidomethylene-N-1,2-Dihydroxypropyl)-EthylenediamineN,N'-Diacetate (Mn-EDTA-ODP)

EDTA-ODP (0.942 g) was dissolved in 200 ml water. Manganese carbonate(0.115 g) was suspended in the reaction mixture and stirred overnight at35° C. Carbon dioxide was released and the mixture was heated to 70° C.The reaction mixture became a soap-like solution, almost transparent.The reaction mixture was then rotary evaporated to dryness, and 1 gsoap-like solid, Mn-EDTA-ODP, was obtained.

The compound prepared in Example 1 is as shown in the structure above.As one skilled in the art will recognize, once armed with the presentdisclosure, the 18 carbon moiety of the acyl chain may be altered, asdesired, using conventional organic chemical techniques. By varying thenumber of carbon atoms in the acyl chains the solubility of theresulting acylated paramagnetic complex, as well as its in vivobiodistribution, may be altered.

EXAMPLE 2

Synthesis of ManganeseN,N'-Bis-(Carboxy-Decylamidomethyl-N-2,3-Dihydroxypropyl)-Ethylenediamine-N,N'-Diacetate(Mn-EDTA-DDP) (Formula I, wherein R₁ is decyl and R₂ is2,3-dihydroxypropyl, n is 0)

Structure ##STR12## Synthetic Route (i) Synthesis of3-Decylamino-1,2-Propanediol (DDP)

The procedures of Ulsperger et al., J. Prakt. Chemie, Vol. 27, pp.195-212 (1965), the disclosures of which are hereby incorporated hereinby reference in their entirety, were substantially followed.Specifically, 15.8 g decylamine (0.1 M) and 7.4 g glycidol (0.1 M) weremixed in 250 ml methanol at 60-80° C. and ref luxed for 10 hours. Themethanol was rotary evaporatated. The product was a semisolid, 23.2 g(yield 100%). After recrystallization with hexane, pure white solid DDP,m.p. 65-67° C. (m.p.70-70.5° C., lit.), was recovered.

(ii) Synthesis ofN,N'-Bis-(Carboxy-Decylamidomethyl-N-2,3-Dihydroxypropyl)-Ethylenediamine-N,N'-DiaceticAcid (EDTA-DDP)

EDTA anhydride 0.005 N (1.28 g) and DDP 0.01 M (2.31 g) were mixedtogether in 100 ml dried methanol. The reaction mixture was stirred at35-40° C. for 12 hours, while the EDTA anhydride particles disappearedand the solution became transparent. The reaction mixture was thenrotary evaporated to dryness, yielding 3.2 g (89%) of a white solid,EDTA-DDP.

Elemental Analysis: C₃₆ H₇₀ N₄ O₁₀ ; Calc. C 60.14; H 9.81; N 7.79;Anal. C 59.04; H 10.10; N 7.54.

(iii) Synthesis of ManganeseN,N'-Bis-(Carboxy-Decylamidomethyl-N-2,3-Dihydroxypropyl)-Ethylenediamine-N,N'-DiaceticAcid (Mn-EDTA-DDP)

Manganese carbonate (0.23 g) and EDTA-DDP (1.44 g) were added to 100 mlwater, and the reaction mixture stirred overnight at 40-45° C. Carbondioxide was released, and the mixture was heated to 70° C., at whichtime the reaction mixture became a soap-like solution, almosttransparent. This was rotary evaporated to dryness, and a soap-likesolid, 1.39 g (89.8% yield) Mn-EDTA-DDP, was obtained.

EXAMPLE 3

Synthesis of ManganeseN,N'-Bis-(Carboxy-Laurylamidomethyl-N-2,3-Dihydroxypropyl)-Ethylenediamine-N,N'-Diacetate(Mn-EDTA-LDP) (Formula I, wherein R₁ is dodecyl, R₂ is2,3-dihydroxypropyl, and n is 0)

Structure ##STR13## Synthetic Route (i) Synthesis of3-Laurylamino-1,2-Dihydroxy-Propane (LDP)

The procedures of Ulsperger et al., J. Prakt. Chemie, Vol. 27, pp.195-212 (1965), the disclosures of which are hereby incorporated hereinby reference in their entirety, were substantially followed.Specifically, 18.54 g laurylamine (0.1 M) and 7.4 g glycidol (0.1 M)were mixed in 150 ml methanol at 60° C. for 5 hours. The mixture wasrefluxed for 1 hour at 70° C. The methanol was then removed by rotaryevaporation. The product was a solid, 15.3 g (59% yield). Afterrecrystallization from hexane, LDP, was recovered as a white crystal,m.p. 75-76° C. (m.p. 76-76.5° C., lit.).

(ii) Synthesis ofN,N'-Bis-(Carboxy-Laurylamidomethyl-N-2,3-Dihydroxypropyl)-Ethylenediamine-N,N'-DiaceticAcid (EDTA-LDP)

EDTA anhydride (2.56 g; 0.01 M) and LDP (5.19 g; 0.02 M) were dissolvedtogether in fresh dried methanol (160 ml). The reaction mixture wasstirred at 35-40° C. for 12 hours, while the EDTA anhydride particlesdisappeared and the solution became transparent. The reaction mixturewas then rotary evaporated to dryness and 7.75 g white solid wasobtained (100% yield), of EDTA-LDP.

Elemental analysis: C₄₀ H₇₈ N₄ O₁₀ ; Calc. C: 61.99 H: 10.14 N: 7.23;Anal. C: 61.50 H: 10.18 N: 7.06.

(iii) Synthesis of ManganeseN,N-Bis-(Carboxy-Laurylamidomethyl-N-2,3-Dihydroxypropyl)-Ethylenediamine-N,N'-Diacetate(Mn-EDTA-LDP)

Manganese carbonate (0.19 g; 0.0016 M) and EDTA-LDP (1.25 g; 0.0016 M)were added to 200 ml water, and the reaction mixture stirred overnightat 40° C. Carbon dioxide was released, and the mixture was heated to 70°C., at which time the reaction mixture became a soap-like solution,almost transparent. This was rotary evaporated to dryness, and 0.92 g ofa soap-like solid, Mn-EDTA-LDP (yield 68.4%), was obtained.

EXAMPLE 4

Synthesis of ManganeseN,N"-Bis-(Carboxyamidomethyl-N-2-Methoxyethylene)-N-Carboxy-Octadecylamidomethyl-Diethylenetriamine-N,N"-Diacetate (Mn-DTPA-OA) (Formula IV, wherein R₁ is octadecyl, R₂ is H,R₃ is 2-methoxyethyl, R₄ is H, A is N, and z is 1)

Structure ##STR14## Synthetic Route (i) Synthesis ofN,N"-Bis(Carboxyamidomethyl-N-(2-Methoxyethyl))-Diethylenetriamine-N,N',N"-TriaceticAcid (DTPA-MEA)

Diethylenetriamine-N,N',N"-triacetic acid (DTPA) (0.79 g) and freshdistilled 2-methoxyethylamine (0.3 g) were mixed in dried methanol (50ml) and stirred overnight. The mixture became transparent. The methanolwas then evaporated and 0.84 g of a white solid, DTPA-MEA, obtained.

(ii) Synthesis ofN,N"-Bis-(Carboxyamidomethyl-N-2-Methoxyethylene)-N'-Carboxy-Octadecylamidomethyl-Diethylenetriamine-N,N"-Diacetic Acid (DTPA-OA-MEA)

Octadecylamine (0.807 g) and DTPA-MEA (1.296 g) were mixed together withN-dimethylforamide (DMF) (30 ml), and added dropwise to a solution ofdicyclohexylcarbodiimide (DCC) in 5 ml DMF at 0-5° C., and stirred for 2hours. The temperature was then raised to 40-45° C. for one additionalhour, after which the reaction was completed. The DMF was thenevaporated off under reduced pressure, the residue diluted with water,and the precipitate filtered out. The water was then evaporated underreduced pressure, yielding 1.5 g of a soap-like material, DTPA-OA-MEA.

(iii) Synthesis of ManganeseN,N"-Nis-(Carboxyamidomethyl-N-2-Methoxyethylene)-N'-Carboxy-Octadecylamidomethyl-Diethylenetriamine-N,N"-Diacetate(Mn-DTPA-OA)

Manganese carbonate (0.25 g) and DTPA-OA-MEA (1.5 g) were mixed with 80ml of water and stirred over night, resulting in a soap-like solution.Another portion of manganese carbonate (0.25 g) was then added andstirred overnight. The small amount of unreacted manganese carbonate wasfiltered off and the sample was evaporated using a rotary evaporator,yielding 1.86 g of a soap-like material, (Mn-DTPA-OA).

EXAMPLE 5

GadoliniumN,N"-Bis-(Carboxyoctadecylamidomethyl-N-2,3-Dihydroxypropyl)-Diethylenetriamine-N,N"-Triacetate(Gd-DTPA-ODP) (Formula I, wherein R₁ is octadecyl, R₂ is2,3-dihydroxypropyl, n is 1)

Structure ##STR15## Synthetic Route (i) Synthesis ofN,N"-Bis-(Carboxyoctadecylamidomethyl-N-2,3-Dihydroxypropyl)-Diethylenetriamine-N,N',N"-Triaceticacid (ODP-DTPA)

ODP (3.43 g) was dissolved in 150 ml dried methanol and heated to 40° C.The anhydride of diethylenetriaminepentaacetic acid (DTPA) (1.79 g) wasadded by stirring, and the mixture stirred overnight. The solutionbecame transparent. The solution was then evaporated and a white solidproduct, ODP-DTPA (5.2 g), obtained.

(ii) Synthesis of GadoliniumN,N"-Bis-(Carboxyoctadecylamidomethyl-N-2,3-Dihydroxypropyl)-Diethylenetriamine-N,N',N"-Triacetate(Gd-DTPA-ODP)

Gadolinium chloride (0.34 g) (containing 28.8% water) was dissolved in20 ml of ethanol, mixed with one gram of ODP-DTPA in 20 ml of ethanol,stirred for 24 hours, and then evaporated to dryness. Ethanol (20 ml)was again added to the mixture, and the mixture again evaporated todryness. This step was repeated three additional times, yielding 1.168 gof Gd-DTPA-ODP.

EXAMPLE 6

Synthesis of FerricN,N"-Bis(Carboxyoctadecylamidomethyl-N-2,3-Dihydroxypropyl)-Diethylenetriamine-N,N',N"-Triacetate(Fe-DTPA-ODP) (Formula I, wherein R₁ is octadecyl, R₂ is2,3-dihydroxypropyl, n is 1)

Structure ##STR16## Synthetic Route

Synthesis of FerricN,N"-Bis(Carboxyoctadecylamidomethyl-N-2,3-Dihydroxypropyl)-Diethylenetriamine-N,N',N"-Triacetate(Fe-DTPA-ODP)

Ferric chloride (0.16 g) was dissolved in 20 ml of ethanol and mixedwith 1 g of ODP-DTPA in 20 ml of ethanol, stirred for 24 hours, andevaporated to dryness. To this was again added 20 ml of ethanol, and themixture evaporated to dryness. This step was repeated an additionalthree times. A green-yellow solid of about 1 g, Fe-DTPA-ODP, wasobtained.

EXAMPLE 7

Synthesis of Manganese1,7-Bis-(Carboxy-Octadecylamidomethyl-N-2,3-Dihydroxypropyl)-1,4,7,10-Tetraazacyclododecane-4,10-Diacetate(Mn-DOTA-ODP) (Formula III, wherein R₁ is octadecyl, R₂ is2,3-dihydroxypropyl, n is 1, and m is 1)

Structure ##STR17## Synthetic Route (i) Synthesis of1,4,7,10-Tetraazacyclododecane-1,4,7,10-Tetraacetic Acid (DOTA)Anhydride

Two g of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid wasmixed with 30 g of acetic anhydride and heated for eight hours. Thereaction mixture was cooled down to room temperature and the precipitatefiltered, resulting in DOTA anhydride.

(ii) Synthesis of1,7,-Bis-(Carboxy-Octadecylamidomethyl-N-2,3-Dihydroxypropyl)-1,4,7,10-Tetraazacyclododecane-4,10-Diaceticacid (DOTA-ODP)

DOTA anhydride (0.74 g) and ODP (1.37 g) were mixed with 50 ml freshdried methanol and stirred overnight. The reaction mixture becametransparent. The methanol was then evaporated off, yielding a whitesolid, DOTA-ODP.

(iii) Synthesis of Manganese1,7-Bis-(Carboxy-Octadecylamidomethyl-N-2,3-Dihydroxypropyl)-1,4,7,10-Tetraazacyclododecane-4,10-Diacetate(Mn-DOTA-ODP)

Manganese carbonate (0.115 g) and DOTA-ODP (1 g) were mixed togetherwith 100 ml water and stirred for two hours, then heated to 40° C., andstirred for an additional two hours. The reaction mixture wasevaporated, and a 1 g soaplike solid, Mn-DOTA-ODP, was obtained.

EXAMPLE 8

Preparation of Liposomal Mn-EDTA-ODP, Mn-DTPA-OA-MEA, Gd-DTPA-ODP,Mn-EDTA-DDP and Mn-EDTA-DDP

Mn-EDTA-ODP was incorporated into small unilamellar liposomes asfollows. Egg phosphatidylcholine (EPC) and cholesterol (8:2 molar ratio)were suspended in chloroform and a 33 percent molar concentration ofMn-EDTA-ODP was added to the solution. The chloroform was thenevaporated under vacuum and the dried lipids and Mn-EDTA-ODP wereresuspended in phosphate buffered saline (PBS). The mixture wastransferred to a cryovial, quench frozen in liquid nitrogen, and thawedfive times. The material was then extruded through an extruder device(Lipex Biomembranes, Vancouver, B.C., Canada) 10 times using a 400 nmdiameter pore size polycarbonate filter to produce 400 nm liposomes. Aportion of the 400 nm liposomes were then extruded through 100 nmdiameter filters 10 times to produce 100 nm liposomes. A portion of the100 nm liposomes were then extruded 10 times through 15 nm filters,producing liposomes of 30 nm size. Previously, it was shown byquasi-elastic light scattering that such extrusions through 400 nmfilters produces liposomes of about 400 nm size, through 100 nm filtersproduces liposomes of about 100 nm size, and through 15 nm filtersproduces liposomes of about 30 nm in size. In a similar fashion, 400 nm,100 nm and 30 nm liposomal Mn-DTPA-OA-MEA, Gd-DTPA-ODP, Mn-EDTA-DDP andMn-EDTA-DDP compounds were also prepared.

EXAMPLE 9

Intravenous lipid emulsions are formulated with a contrast agent of theinvention to provide an emulsified preparation comprising the contrastagent of the invention following the techniques and using theingredients described in Modern Pharmaceutics, pp. 505-507, GilbertBaker and Christopher Rhodes, eds., Marcel Dekker Inc., New York, N.Y.(1990). Specifically, the following emulsions are prepared:

Example 9A: soybean oil 10%, egg phosphatidylcholine (EPC) 1.2%,glycerol 2.25%, 100 ml of water.

Example 9B: soybean oil 20%, EPC 1.2%, glycerol 2.25%, 100 ml of water.

Example 9C: soybean oil 5%, safflower oil 5%, EPC 1.2%, glycerol 2.5%,100 ml water.

Example 9D: cottonseed oil 15%, soybean phospholipid 1.2%, and sorbitol5%.

EXAMPLE 10

Synthesis of Bi-Mn-EDTA-DDP (LDP,ODP) (Formula V, wherein R₁ isoctadecyl, R₂ is 2,3-dihydroxypropyl, R₃ is ethylene, and m is 0)

Structure ##STR18## Synthetic Route (i) Synthesis of N,N'Di-s,3-Dihydroxypropyl-Ethylenedimine (Di-DPEA)

Ethylenediamine (6 g) was dissolved in methanol (70 ml), and heated to60° C. Glycidol (14.8 g) diluted with methanol (30 ml), added dropwiseinto the boiling solution of ethylenediamine, for 45 minutes. Themixture was stirred and refluxed for two additional hours. The methanolwas evaporated by a rotary evaporator, resulting in 20 g of Di-DPEA.

(ii) Synthesis of Bi-EDTA-DDP

Two grams Di-DPEA was dissolved in 30 ml dried methanol, added dropwise,and stirred thoroughly. Next, 5.1 g EDTA anhydride and 100 ml driedmethanol was added to the mixture over one hour at room temperature, andthe mixture continuously stirred for 3 hours at room temperature. DDP(4.7 g) was added into the reaction mixture, and the mixture stirred forfour additional hours. The reaction temperature was then raised to 50°C., the mixture stirred for one hour, and the solvent evaporated,resulting in 11.4 g solid Bi-EDTA-DDP.

(iii) Synthesis of Bi-Mn-EDTA-DDP

Bi-EDTA-DDP (5.9 g) was dissolved in 100 ml water, and manganesecarbonate (1.2 g) added. The mixture was stirred overnight, and thenheated to 70° C. and stirred for an additional hour. The water wasevaporated off, yielding 6 g Bi-Mn-EDTA-DDP.

As the structure shown above for Example 10 reveals, the compoundBi-Mn-EDTA-DDP contains a chelating unit that is able to chelate morethan a single paramagnetic ion. Although this compound is shownchelating only two Mn ions, it may, if desired, be prepared to chelatemore than one of paramagnetic ions in one molecule, for example, Mn⁺²and Fe⁺², Gd⁺³ and Fe⁺³, Gd⁺³ and Mn⁺², and Fe⁺³ and Fe⁺².

EXAMPLE 11

One gram of human serum albumin, obtained from pooled human serum, wasmixed with 10 mg of EDTA-DDP in 10 cc of normal saline. The mixture wassonicated with a Heat Systems probe Sonicator (Heat Systems Probes,Farmingdale, N.Y.) at level 4 for 1 minute. The material was then cooledto 4° C. and, after 48 hours, 2.5 mg of MnCl₂ was added to thepreparation. The preparation was then dialyzed against normal saline for48 hours, generating Mn-EDTA-DDP suspended in (non-covalently bound to)albumin.

EXAMPLE 12

The procedures of Example 11 were substantially followed, except thatinstead of sonication, the albumin and Mn-EDTA-DDP were heated to atemperature of 100° C. for two minutes.

EXAMPLE 13

The procedures of Example 12 were substantially followed, except thatthe albumin and Mn-EDTA-DDP were heated to a temperature of 75° C. for60 minutes.

EXAMPLE 14

Liposomes prepared in accordance with Example 8 incorporatingMn-EDTA-DDP in the membrane bilayer were subjected to a Microfluidizer(Microfluidics, Newton, Mass.). Specifically, the liposomes were passed10 times through the microfluidizer at a pressure of 16,000 psi and aflow rate of 450 ml/minute. The resulting liposomes had a mean averagesize of 30-40 nm, which was verified by Quasi Elastic Light Scattering(QEL).

EXAMPLE 15

For comparison to contrast agents of the invention, solutions ofmanganese chloride and manganese chloride liposomes were prepared.Specifically, the MnCl₂ liposomes were prepared by resuspending driedlipids 8:2 EPC/cholesterol in an aqueous solution of manganese chloride.Different concentration solutions of MnCl₂ ranging from 10 to 500millimolar manganese were used to make the MnCl₂ liposomes. Unentrappedmanganese was removed by exhaustive dialysis.

EXAMPLE 16

Synthesis of ManganeseN,N'-Bis-(Carboxy-Octadecylamidomethyl-N-2,3-Dihydroxypropyl)-Cyclohexane-1,2-Diamino-N,N'-Diacetate(Mn-CHTA-ODP) (Formula II, wherein R₁ is octadecyl, R₂ is2,3-dihydroxypropyl, B is cyclohexyl)

Structure ##STR19## Synthetic Route (i) Synthesis ofCyclohexane-1,2-Diamino-N,N,N',N'-Tetraacetic Acid (CHTA) Anydride

Cyclohexane-1,2-diamino-N,N,N',N'-tetraacetic acid (3.46 g) was mixedwith acetic anhydride (30 g), and heated for 8 hours. The reactionmixture was cooled to room temperature, and the precipitate filteredout, yielding cyclohexane-1,2-diamino-N,N,N',N'-tetraacetic acidanhydride.

(ii) Synthesis ofN,N',-Bis-(Carboxy-Octadecylamidomethyl-N-2,3-Dihydroxypropyl)-Cyclohexane-1,2-Diamino-N,N'-Diaceticacid(CHTA-ODP)

CHTA anhydride (3.1 g) and ODP (6.86 g) was mixed with 100 ml freshdried methanol, and stirred overnight. The reaction mixture becametransparent. The methanol was then evaporated off, resulting in a whitesolid, CHTA-ODP.

(iii) Synthesis of ManganeseN,N'-Bis-(Carboxy-Octadecylamidonlethyl-N-2,3-Dihydroxypropyl)-Cyclohexane-1,2-Diamino-N,N'-Diacetate(Mn-CHTA-ODP)

Manganese carbonate (0.6 g) and CHTA-ODP (5 g) was mixed together with100 ml water, stirred for 2 hours, and then heated to 40° C. The mixturewas stirred for an additional two hours, and the water evaporated,yielding 5 g of a soap like solid, Mn-CHTA-ODP.

EXAMPLE 17

In Vitro Relaxivity of Liposomal Mn-EDTA-ODP, Mn-DTPA-OA-MEA,Gd-DTPA-ODP, Mn-EDTA-DDP and Mn-EDTA-DDP

Liposomal contrast agents of the invention, prepared in accordance withExample 8, were serially diluted from a stock solution of knownconcentration. Diluted concentrations for testing were held constant at0.5 mM, 0.25 mM, 0.125 mM, 0.100 mM, 0.05 mM, and 0.025 mM,respectively. Samples were scanned on a Toshiba MRT 50A 0.5 Tesla (21.3MHz) clincal magnet equipped with a QD head coil (Toshiba MRI scanner,Nasu, Japan). Signal intensities for resulting scans were thenstatistically analyzed using a computer curve fitting program (Fit All,MTR Software, version 1.1). Resulting relaxivities were regressedagainst the concentration to determine R1 (1/T1 mmol sec⁻¹) and R₂ (1/T1mmol sec⁻¹). The results were compared with similar scans for othercompounds not within the scope of the present invention. Specifically,as a comparison for the contrast agents of the invention, 0.5 Teslascans were made of Gd-DTPA (no liposome), Mn-EDTA-MEA (no liposome),Mn-EDTA-MEA (incorporated into a liposome of 0.1 micron), and phosphatebuffered saline (PBS).

The results are shown in Table I below. As shown in Table I, thecontrast agents of the invention have excellent relaxivity. Therelaxivity is greatest for the smallest (30 nm) liposomes containingMn-EDTA-DDP.

                  TABLE I                                                         ______________________________________                                        Relaxivity of Contrast Agents at 0.5 Tesla                                    Sample           R1         R2                                                ______________________________________                                        PBS              0.300 ± 0.30                                                                          0.395 ± 0.169                                  Gd-DTPA          4.68 ± 0.279                                                                           5.17 ± 0.148                                  Mn-EDTA-MEA      3.12 ± 0.124                                                                           5.61 ± 0.011                                  Gd-DTPA-ODP liposomes                                                                          3.427 ± 0.141                                                                         4.190 ± 0.087                                  0.1 micron                                                                    Mn-EDTA-MEA liposomes                                                                          0.941 ± 0.045                                                                          1.12 ± 0.117                                  0.1 micron                                                                    Mn-DTPA-MEA-OA liposomes                                                                       1.216 ± 0.0827                                                                        1.631 ± 0.211                                  0.4 micron                                                                    Mn-EDTA-ODP liposomes                                                                          7.77 ± 0.742                                                                          11.44 ± 0.83                                   0.4 micron                                                                    Mn-EDTA-ODP liposomes                                                                          17.44 ± 0.97                                                                          23.6 ± 1.82                                    0.1 micron                                                                    Mn-EDTA-ODP liposomes                                                                          31.77 ± 1.99                                                                          35.0 ± 1.76                                    0.03 micron                                                                   Mn-EDTA-LDP liposomes                                                                          18.39 ± 0.231                                                                         22.46 ± 0.687                                  0.1 micron                                                                    Mn-EDTA-DDP liposomes                                                                          5.73 ± 0.195                                                                           7.22 ± 0.100                                  0.4 micron                                                                    Mn-EDTA-DDP liposomes                                                                          30.27 ± 1.15                                                                          36.69 ± 1.26                                   0.1 micron                                                                    Mn-EDTA-DDP liposomes                                                                          37.4 ± 1.12                                                                            53.2 ± 0.228                                  0.03 micron                                                                   ______________________________________                                    

In all liposome examples in Table I, the lipid concentration is 200 mM,and all liposomes are composed of 80 mole percent egg phosphatidylcholine (EPC) and 20 mole percent cholesterol. Also, for each liposomeand compound combination (e.g., Mn-EDTA-DDP liposomes) the liposomescomprise 33 mole percent of the compound (e.g. Mn-EDTA-DDP) and 67 molepercent lipid (8:2 EPC/cholesterol).

In Table I, R1 and R2 refer to 1/T1 and 1/T2 per millimole ofparamagnetic ion per sec⁻¹, except for phosphate buffered saline (PBS),which refers to 1/T1 and 1/T2 for comparision.

Gd-DTPA, Mn-EDTA-MEA, Mn-EDTA-MEA liposomes, and PBS are all comparativeexamples. Gd-DTPA and Mn-EDTA-MEA are complexes without liposomes.Mn-EDTA-MEA liposomes refers to the complex entrapped within liposomes.For all others liposome examples, the respective complexes areincorporated into membranes of liposomes.

As Table I clearly illustrates, the contrast agents of the inventionshow high relaxivity.

EXAMPLE 18

Stability of Liposomal Mn-EDTA-ODP

Stability experiments were carried out with liposomal Mn-EDTA-ODPcontrast agents of the invention, prepared in accordance with Example 8.To carry out the experiments, Mn-EDTA-ODP liposomes were placed withindialysis tubing with a 500 molecular weight cutoff (Sprectrum Medical,Los Angeles, Calif.) containing either PBS or PBS and 50% human serum.Dialysis tubing was suspended within a 500 ml beaker containing PBSwhich was placed into a shaking water bath maintained at 40° C. Two mlsamples of each preparation were obtained from the dialysis tubing at 0,12, and 24 hours. Samples were analyzed for Mn⁺² concentration by aspectrophotometric assay. PBS within the beakers was changed ever 8hours.

The results are shown in Table II. The low level of change in eachsample indicates a high stability of the contrast agents of theinvention. The high serum stability, in particular, sets the contrastagents of the invention apart from many of the contrast agents knownheretofor.

                  TABLE II                                                        ______________________________________                                        Serum stability of Mn-EDTA-ODP Liposomes                                      Measured In Percentage Manganese Retained                                     Liposome Diameter                                                                          Initial   12 hours 24 hours                                      ______________________________________                                        0.1 μ + PBS                                                                             100       85.29    84.45                                         0.4 μ + PBS                                                                             100       97.90    95.39                                         0.1 μ + 50% serum                                                                       100       91.18    96.22                                         0.4 μ + 50% serum                                                                       100       96.22    96.22                                         ______________________________________                                    

EXAMPLE 19

In Vitro Relaxivity of Mn-EDTA-DDP and Mn-EDTA-DDP Albumin Suspensions

Mn-EDTA-DDP and Mn-EDTA-DDP albumin suspensions (contrast agents withinthe scope of the invention) were prepared in accordance with Example 11,except that water instead of saline was used. The samples scanned by NMRusing a 0.5 Tesla (21.3 MHz) Toshiba MRI scanner (Nasu, Japan) todetermine relaxivity. The results were compared with similar scans forother compounds not within the scope of the invention. Specifically,scans were made of contrast agent of the invention, Mn-EDTA-DDP,Mn-EDTA-DDP albumin suspensions (both heated to 55° C., and unheated),and compared with scans of PBS, Gd-DTPA, MnCl₂, and MnCl₂ albuminsuspensions. MnCl₂, and the MnCl₂ liposomes were prepared in accordancewith Example 15.

The results are shown in Table III. Comparing the relaxivity of thealbumin Mn-EDTA-DDP to the relaxivity of the Mn-EDTA-DDP alone, there isa significant improvement in relaxivity for the contrast agent withalbumin. Not intending to be bound by any theory of operation, theimprovement in relaxivity of Mn-EDTA-DDP with albumin is believed toresult from albumin binding with the contrast agent. This binding islikely non-covalent and due to Van der Waals forces, representing anattraction between the acyl chains of the Mn-EDTA-DDP and thehydrophobic domains of the albumin molecule. The data also show thatalbumin with manganese causes no similar improvement in relaxivity,i.e., the relaxivity of manganese plus albumin is similar to manganeseion alone. Whether or not the albumin is heated appears to have littleeffect on the increase in relaxivity of Mn-EDTA-DDP.

                  TABLE III                                                       ______________________________________                                        In Vitro Relaxivity of Manganese and Mn-EDTA-DDP                              With and Without Albumin 0.5 Tesla                                            Sample           R1        R2                                                 ______________________________________                                        Albumin w/MnCl.sub.2                                                                           8.39 ± 0.446                                                                         34.18 ± 0.689                                   Albumin          24.6 ± 0.375                                                                         37.0 ±  1.21                                    Mn-EDTA-DDP                                                                   Mn-EDTA-DDP-Albumin                                                                            23.3 ± 0.593                                                                          34.1 ± 0.481                                   (Heated to 55° C.)                                                     MN-EDTA-DDP      9.83 ± 0.332                                                                         15.20 ± 0.393                                   MnCl.sub.2       8.73 ± 0.928                                                                         39.45 ± 0.515                                   Gd-DTPA          4.58 ± 0.143                                                                         5.41 ± 0.65                                     1.0 mM                                                                        ______________________________________                                    

EXAMPLE 20

In Vivo Efficacy of Mn-EDTA-ODP and Mn-EDTA-DDP Liposomes

Mn-EDTA-ODP and Mn-EDTA-DDP liposomes of both 30 nm and 100 nm (contrastagents within the scope of the invention) were prepared in accordancewith Example 8, injected intraveneously via a tail vein injection intorats bearing hepatic tumors (C5 clonal derivative epithelioidneoplasms), and the rats imaged using a 1.5 Tesla GE Signa ClinicalMagnet equipped with a linear knee coil. Animals wre anesthesized with a10:1 mixture v/v of ketamine (100 mg/ml) and acepoumozine (10 mg/ml)prior to imaging. Imaging parameters were: TR=250; TE=12;Matrix=256×192; NEX=8; FOV 16 cm; Slice Thickness=3 mm; Slice Gap=1 mm.Images were taken in the coronal plane, mapped off an axial scout image.For comparision, rats were also injected with MnCl₂, and MnCl₂liposomes, prepared in accordance with Example 15.

The results are shown in Tables IV A-D. The data for Mn-EDTA-ODP 30 nmliposomes is shown in Table IV A. As the data indicates, the Mn-EDTA-ODPliposomal contrast agents are highly effective. Also, as shown by TablesIV B, C and D, Mn-EDTA-DDP liposomes are much more effective than eitherfree MnCl₂ or MnCl₂ liposomes. Hepatic enhancement was much morespecific with the Mn-EDTA-DDP 100 nm liposomes than for either MnCl₂ orMnCl₂ liposomes.

                  TABLE IV A                                                      ______________________________________                                        In Vivo Efficacy of Mn-EDTA-ODP Liposomes                                     (30 nm diameter)                                                                           Rat 1     Rat 2   Rat 3   Rat 4                                       ID      40 μmol/kg                                                                           100 μmol/kg                                                                        100 μmol/kg                                                                        200 μmol/kg                         ______________________________________                                        Pre  Liver & 232 ± 26                                                                             218 ± 20                                                                           217 ± 23                                                                           172 ± 23                                 Muscle  130 ± 22                                                                             110 ± 18                                                                           103 ± 24                                                                           103 ± 16                                 Noise    27 ± 11                                                                              27 ± 11                                                                            37 ± 15                                                                            37 ± 15                                 S/N                                                                           Ratio                                                                         Liver & 8.6       8.1     5.9     4.6                                         Muscle  4.8       4.1     2.8     2.8                                    Post Liver & 435 ± 57                                                                             447 ± 35                                                                           515 ± 52                                                                           329 ± 49                                 Muscle   98 ± 16                                                                             141 ± 19                                                                           225 ± 18                                                                           200 ± 15                                 Noise   23 ± 9 23 ± 9                                                                              29 ± 11                                                                            29 ± 11                                 S/N                                                                           Ratio                                                                         Liver & 18.9      19.4    17.8    11.3                                        Muscle  4.3       6.1     7.8     6.9                                    ______________________________________                                    

In Table IV A, imaging was preformed with one rat at each dose. S/Ndenotes signal to noise ratio.

                  TABLE IV B                                                      ______________________________________                                        In Vivo Efficacy of Mn-EDTA-DDP Liposomes                                     Percent Liver Enhancement                                                                                          Mn-EDTA-DDP                                                      MnCl.sub.2 Liposomes                                                                       Liposomes                                                        (100 nm      (100 nm)                                        MnCl.sub.2       diameter)    diameter)                                Dosage        delayed        delayed      delayed                             μM/kg                                                                             post   post *    post post *  post post *                              ______________________________________                                        0.5    0      0         NA   NA      26   26                                  1.0    0      0         18.3 18.9    34   31                                  2.5    25     29.4      36   43      44   42                                  5.0    43     21        62.4 53.2    88   86.5                                10     81     61        84.1 74.2    100  92                                  ______________________________________                                    

In Table IV B, the "*" denotes a 30 minutes delay in imaging. Also, NAdenotes that imaging was not done at the indicated dosage. The liposomesemployed were composed of 80 mole percent egg phosphatidylcholine (EPC)and 20 mole percent cholesterol. With the Mn-EDTA-DDP liposomes, therewas a 1:3 molar ratio of Mn-EDTA-DDP to lipid in the liposomes (lipidwas 8:2 EPC/cholesterol). The data was obtained from one rat imaged ateach dose.

                  TABLE IV C                                                      ______________________________________                                        In Vivo Efficacy of Mn-EDTA-DDP Liposome                                      Tumor Contrast to Noise                                                                                            Mn-                                                                           EDTA-DDP                                                                      Liposomes                                                         MnCl.sub.2  (100 nm                                  Dosage   MnCl.sub.2      Liposomes   diameter)                                μM/kg pre    post     pre  post   pre  post                                ______________________________________                                        0.5      28     19       NA   NA     28   38.3                                1.0      37.5   23       NT   NT     21   29.4                                2.5      13.1   17.9     35.5 51.5   12.5 67                                  5.0      21.3   28.3     26.5 73.0   NT   NT                                  10.0     9.3    29.2     27.5 80.0   7.8  56                                  ______________________________________                                    

In Table IV C, NT denotes that no tumors were detected, and NA denotesthat imaging not done at the indicated dosage.

                  TABLE IV D                                                      ______________________________________                                        In Vivo Efficacy of Mn-EDTA-DDP Liposomes                                     Tumor Contrast To Noise                                                       (30 minute delay)                                                                                 MnCl.sub.2                                                                             Mn-EDTA-DDP                                                          Liposomes                                                                              Liposomes                                        Dosage              (100 nm  (100 nm                                          μM/kg                                                                              MnCl.sub.2  diameter)                                                                              diameter)                                        ______________________________________                                        0.5     12          NA       49.2                                             1.0     16          NT       37.6                                             2.5     35.9        40       50                                               5.0     31.3        62.0     NT                                               10.0    29.6        60.0     59                                               ______________________________________                                    

In Table IV D, NT denotes that no tumors were detected, and NA denotesthat imaging not done at the indicated dosage.

EXAMPLE 21

In Vivo Toxicity of Mn-EDTA-DDP and Mn-EDTA-DDP Liposomes

Outbred ICR mice (Harlan Sprague Dawley, Indianapolis, Ind.) wereinjected intraveneously via a tail vein injection with various doses ofMn-EDTA-DDP and Mn-EDTA-DDP liposomes, prepared in accordance withExample 8, and the LD50 measured. As a comparision, the mice were alsoinjected with MnCl₂ and MnCl₂ liposomes.

The results are shown in Table V. As Table V reveals, liposomes bearingMn-EDTA-DDP are the least toxic of any of the compounds tested. UsingMn-EDTA-DDP liposomes, the LD50 was greater than 1,062 micromoles ofmanganese per kg. This confers a therapeutic index of more than 400:1,asssuming an imaging dose of 2.5 μmol/kg (more than adequate forimproving liver to tumor contrast). At a dose of 1062 μmol/kg,Mn-EDTA-DDP liposomes all mice survived and had similar activity scoresas mice receiving normal saline.

                  TABLE V                                                         ______________________________________                                        In Vivo Toxicity Testing                                                                         Interpolated LD50s                                         Agent              (μmole/kg)                                              ______________________________________                                        MnCl.sub.2         250                                                        MnCl.sub.2 Liposomes                                                                             700                                                        Mn-EDTA-DDP        240                                                        Mn-EDTA-DDP in Liposomes                                                                         >1062                                                      ______________________________________                                    

In Table V, liposomes denotes manganese chloride salt entrapped in 100nm diameter liposomes comprised of 8:2 EPC/cholesterol. Also,Mn-EDTA-DDP in liposomes refers to 100 nm liposomes comprised of 1:3Mn-EDTA-DDP to lipid (where the lipid is 8:2 EPC/cholesterol).

What is claimed is:
 1. A contrast agent for magnetic resonance imagingcomprising a paramagnetic ion in combination with a compound of theformula ##STR20## wherein: each R₁ is, independently, a substituted orunsubstituted C₇ -C₃₀ straight chain or cyclic compound;each R₂ is,independently, a substituted or unsubstituted C₁ -C₃₀ straight chain orcyclic compound which may be internally interrupted by O, NH, NR₃, or S,where R₃ is a C₁ -C₃ alkyl; each m is 1 to 2; and n is 1 to
 20. 2. Acontrast agent of claim 1 wherein R₁ is an unsubstituted C₇ -C₃₀ alkyl.3. A contrast agent of claim 2 wherein R₁ is an unsubstituted C₈ -C₁₈alkyl.
 4. A contrast agent of claim 1 wherein at least one of said R₂groups is a substituted or unsubstituted C₁ -C₃₀ straight chain orcyclic compound which is internally interrupted by O, NH, NR₃ or S.
 5. Acontrast agent of claim 4 wherein at least one of said R₂ groups is asubstituted or unsubstituted C₂ -C₁₂ straight chain or cyclic compoundwhich is internally interrupted by O, NH, NR₃ or S.
 6. A contrast agentof claim 1 wherein n is 1 to
 2. 7. A contrast agent of claim 1 whereinR₁ is octadecyl, R₂ is 2,3-dihydroxypropyl, m is 1, and n is
 1. 8. Acontrast agent of claim 1 wherein the paramagnetic ion comprises an ionselected from the group consisting of Cr⁺³, Co⁺², Mn⁺², Ni⁺², Fe⁺³,Fe⁺², La⁺³, Cu⁺², Gd⁺³, Ce⁺³, Tb⁺³, Pr⁺³, Dy⁺³, Nd⁺³, Ho⁺³, Pm⁺³, Er⁺³,Sm⁺³, Tm⁺³, Eu⁺³, Yb⁺³ and Lu⁺³.
 9. A contrast agent of claim 8 whereinthe paramagnetic ion comprises an ion selected from the group consistingof Mn⁺², Fe⁺³ and Gd⁺³.
 10. A contrast agent of claim 9 wherein theparamagnetic ion is Mn⁺².
 11. A contrast agent of claim 7 wherein theparamagnetic ion is Mn⁺².
 12. A contrast agent of claim 1 furthercomprising a lipid compound.
 13. A contrast agent of claim 12 whereinthe lipid compound comprises a lipid selected from the group consistingof cholesterols, phosphatidylcholines, phosphatidylethanolamines,phosphatidylserines, phosphatidylglycerols, phosphatidic acids,phosphatidylinositols, phospholipids, lysolipids, fatty acids,sphingomyelin, glycosphingolipids, glucolipids, glycolipids,sulphatides, lipids with ether and ester-linked fatty acids andpolymerizable lipids.
 14. A contrast agent of claim 12 wherein the lipidcompound is in the form of a lipid emulsion, liposome, or micelle, or acombination thereof.
 15. A contrast agent of claim 1 further comprisinga suspending agent.
 16. A contrast agent of claim 15 wherein thesuspending agent comprises a suspending agent selected from the groupconsisting of polyethylene glycol, lactose, mannitol, sorbitol, ethylalcohol, glycerin, lecithin, polyoxyethylene sorbitan monoleate,sorbitan monoleate and albumin.
 17. A method of providing an image of aninternal region of a patient comprising (i) administering to the patienta contrast agent of claim 1, and (ii) scanning the patient usingmagnetic resonance imaging to obtain visible images of the region.
 18. Amethod for diagnosing the presence of diseased tissue in a patientcomprising (i) administering to the patient a contrast agent of claim 1,and (ii) scanning the patient using magnetic resonance imaging to obtainvisible images of any diseased tissue in the patient.
 19. A contrastagent of claim 5 wherein at least one of said R₂ groups is a substitutedor unsubstituted C₂ -C₆ straight chain or cyclic compound which isinternally interrupted by O, NH, NR₃ or S.
 20. A contrast agent of claim19 wherein at least one of said R₂ groups is a substituted orunsubstituted C₂ -C₆ alkyl group which is internally interrupted by O,NH, NR₃ or S.
 21. A contrast agent of claim 20 wherein at least one ofsaid R₂ groups is a substituted or unsubstituted C₂ -C₆ alkyl groupwhich is internally interrupted by O.
 22. A contrast agent of claim 21wherein at least one of said R₂ groups is unsubstituted.
 23. A contrastagent of claim 1 wherein each of said R₂ groups is, independently, asubstituted or unsubstituted C₁ -C₃₀ straight chain or cyclic compoundwhich is internally interrupted by O, NH, NR₃ or S.
 24. A contrast agentof claim 23 wherein each of said R₂ groups is, independently, asubstituted or unsubstituted C₂ -C₁₂ straight chain or cyclic compoundwhich is internally interrupted by O, NH, NR₃ or S.
 25. A contrast agentof claim 24 wherein each of said R₂ groups is, independently, asubstituted or unsubstituted C₂ -C₆ straight chain or cyclic compoundwhich is internally interrupted by O, NH, NR₃ or S.
 26. A contrast agentof claim 25 wherein each of said R₂ groups is, independently, asubstituted or unsubstituted C₂ -C₆ alkyl group which is internallyinterrupted by O, NH, NR₃ or S.
 27. A contrast agent of claim 26 whereineach of said R₂ groups is, independently, a substituted or unsubstitutedC₂ -C₆ alkyl group which is internally interrupted by O.
 28. A contrastagent of claim 27 wherein each of said R₂ groups is unsubstituted.
 29. Acontrast agent of claim 1 wherein at least one of said R₂ groups isuninterrupted.
 30. A contrast agent of claim 29 wherein at least one ofsaid R₂ groups is a substituted or unsubstituted uninterrupted C₂ -C₁₂straight chain or cyclic compound.
 31. A contrast agent of claim 30wherein at least one of said R₂ groups is a substituted or unsubstituteduninterrupted C₂ -C₆ straight chain or cyclic compound.
 32. A contrastagent of claim 31 wherein at least one of said R₂ groups is asubstituted or unsubstituted uninterrupted C₂ -C₆ alkyl group.
 33. Acontrast agent of claim 32 wherein at least one of said R₂ groups is asubstituted or unsubstituted uninterrupted C₂ -C₆ alkyl group.
 34. Acontrast agent of claim 33 wherein at least one of said R₂ groups is anuninterrupted C₂ -C₆ alkyl group substituted with OH.
 35. A contrastagent of claim 1 wherein:at least one of said R₂ groups is --CH₂(CHOH)_(n) CH₂ OR₄, --CH₂ --(CH₂)_(m) --X--R₄, --(CH₂ CH₂ X)_(m) R₄ or--(CHOH)_(m) --OR₄ ; X is O, NH, NR₃ or S, where R₃ is C₁ -C₃ alkyl; R₄is --(CH₂)_(m) --CH₃ ; and each m is independently an integer from 0 toabout
 26. 36. A contrast agent of claim 35 wherein each of said R₂groups is --CH₂ (CHOH)_(n) CH₂ OR₄, --CH₂ --(CH₂)_(m) --X--R₄, --(CH₂CH₂ X)_(m) R₄ or --(CHOH)_(m) --OR₄.
 37. A contrast agent of claim 36wherein X is O.
 38. A contrast agent for magnetic resonance imagingcomprising a paramagnetic ion in combination with a compound of theformula ##STR21## wherein: each R₁ is, independently, a substituted orunsubstituted C₇ -C₃₀ straight chain or cyclic compound;each R₂ is apolyethylene glycol substituent or a substituted or unsubstituted C₁-C₃₀ straight chain or cyclic compound which may be internallyinterrupted by O, NH, NR₃ or S; R₃ is C₁ -C₃ alkyl; each m is 1 to 2;and n is 1 to 20;provided that at least one of R₂ is a polyethyleneglycol substituent.
 39. A contrast agent of claim 38 wherein each R₂ isindependently a polyethylene glycol substituent.
 40. A contrast agentfor magnetic resonance imaging comprising, in combination with aparamagnetic ion and a lipid compound, a compound of the formula##STR22## wherein: each R₁ is, independently, a substituted orunsubstituted C₇ -C₃₀ straight chain or cyclic compound;each R₂ is,independently, a substituted or unsubstituted C₁ -C₃₀ straight chain orcyclic compound which may be internally interrupted by O, NH, NR₃ or S,where R₃ is C₁ -C₃ alkyl; each m is 1 to 2; and n is 1 to
 20. 41. Acontrast agent of claim 40 wherein the lipid compound is selected fromthe group consisting of cholesterols, phosphatidylcholines,phosphatidylethanolamines, phosphatidylserines, phosphatidylglycerols,phosphatidic acids, phosphatidylinositols, phospholipids, lysolipids,fatty acids, sphingomyelin, glycosphingolipids, glucolipids,glycolipids, sulphatides, lipids with ether and ester-linked fatty acidsand polymerizable lipids.
 42. A contrast agent of claim 41 wherein thelipid compound is in the form of a lipid emulsion, liposome or micelle,or a combination thereof.
 43. A contrast agent for magnetic resonanceimaging comprising, in combination with a paramagnetic ion and asuspending agent, a compound of the formula ##STR23## wherein: each R₁is, independently, a substituted or unsubstituted C₇ -C₃₀ straight chainor cyclic compound;each R₂ is, independently, a substituted orunsubstituted C₁ -C₃₀ straight chain or cyclic compound which may beinternally interrupted by O, NH, NR₃ or S, where R₃ is C₁ -C₃ alkyl;each m is 1 to 2; and n is 1 to
 20. 44. A contrast agent of claim 43wherein the suspending agent is selected from the group consisting ofpolyethylene glycol, lactose, mannitol, sorbitol, ethyl alcohol,glycerin, lecithin, polyoxyethylene sorbitan monoleate, sorbitanmonoleate and albumin.
 45. A compound of the formula ##STR24## wherein:each R₁ is, independently, a substituted or unsubstituted C₇ -C₃₀straight chain or cyclic compound;each R₂ is, independently, asubstituted or unsubstituted C₁ -C₃₀ straight chain or cyclic compoundwhich may be internally interrupted by O, NH, NR₃ or S, where R₃ is C₁-C₃ alkyl; each m is 1 to 2; and n is 1 to
 20. 46. A compound of claim45 wherein R₁ is an unsubstituted C₇ -C₃₀ alkyl.
 47. A compound of claim46 wherein R₁ is an unsubstituted C₈ -C₁₈ alkyl.
 48. A compound of claim45 wherein at least one of said R₂ groups is a substituted orunsubstituted C₁ -C₃₀ straight chain or cyclic compound which isinternally interrupted by O, NH, NR₃ or S.
 49. A compound of claim 48wherein at least one of said R₂ groups is a substituted or unsubstitutedC₂ -C₁₂ straight chain or cyclic compound which is internallyinterrupted by O, NH, NR₃ or S.
 50. A compound of claim 49 wherein atleast one of said R₂ groups is a substituted or unsubstituted C₂ -C₆straight chain or cyclic compound which is internally interrupted by O,NH, NR₃ or S.
 51. A compound of claim 50 wherein at least one of said R₂groups is a substituted or unsubstituted C₂ -C₆ alkyl group which isinternally interrupted by O, NH, NR₃ or S.
 52. A compound of claim 51wherein at least one of said R₂ groups is a substituted or unsubstitutedC₂ -C₆ alkyl group which is internally interrupted by O.
 53. A compoundof claim 52 wherein at least one of said R₂ groups is unsubstituted. 54.A compound of claim 45 wherein each of said R₂ groups is, independently,a substituted or unsubstituted C₁ -C₃₀ straight chain or cyclic compoundwhich is internally interrupted by O, NH, NR₃ or S.
 55. A compound ofclaim 54 wherein each of said R₂ groups is, independently, a substitutedor unsubstituted C₂ -C₁₂ straight chain or cyclic compound which isinternally interrupted by O, NH, NR₃ or S.
 56. A compound of claim 55wherein each of said R₂ groups is, independently, a substituted orunsubstituted C₂ -C₆ straight chain or cyclic compound which isinternally interrupted by O, NH, NR₃ or S.
 57. A compound of claim 56wherein each of said R₂ groups is, independently, a substituted orunsubstituted C₂ -C₆ alkyl group which is internally interrupted by O,NH, NR₃ or S.
 58. A compound of claim 57 wherein each of said R₂ groupsis, independently, a substituted or unsubstituted C₂ -C₆ alkyl groupwhich is internally interrupted by O.
 59. A compound of claim 58 whereineach of said R₂ groups is unsubstituted.
 60. A compound of claim 45wherein at least one of said R₂ groups is uninterrupted.
 61. A compoundof claim 60 wherein at least one of said R₂ groups is a substituted orunsubstituted uninterrupted C₂ -C₁₂ straight chain or cyclic compound.62. A compound of claim 61 wherein at least one of said R₂ groups is asubstituted or unsubstituted uninterrupted C₂ -C₆ straight chain orcyclic compound.
 63. A compound of claim 62 wherein at least one of saidR₂ groups is a substituted or unsubstituted uninterrupted C₂ -C₆ alkylgroup.
 64. A compound of claim 63 wherein at least one of said R₂ groupsis a substituted or unsubstituted uninterrupted C₂ -C₆ alkyl group. 65.A compound of claim 64 wherein said substituent is OH.
 66. A compound ofclaim 45 wherein:at least one of said R₂ groups is --CH₂ (CHOH)_(n) CH₂OR₄, --CH₂ --(CH₂)_(m) --X--R₄, --(CH₂ CH₂ X)_(m) R₄ or --(CHOH)_(m)--OR₄ ; X is O, NH, NR₃ or S, where R₃ is C₁ -C₃ alkyl; R₄ is--(CH₂)_(m) --CH₃ ; and each m is independently an integer from 0 toabout
 26. 67. A compound of claim 66 wherein each of said R₂ groups is--CH₂ (CHOH)_(n) CH₂ OR₄, --CH₂ --(CH₂)_(m) --X--R₄, --(CH₂ CH₂ X)_(m)R₄ or --(CHOH)_(m) --OR₄.
 68. A compound of claim 67 wherein X is O. 69.A compound of the formula ##STR25## wherein: each R₁ is, independently,a substituted or unsubstituted C₇ -C₃₀ straight chain or cycliccompound;each R₂ is a polyethylene glycol substituent or a substitutedor unsubstituted C₁ -C₃₀ straight chain or cyclic compound which may beinternally interrupted by O, NH, NR₃ or S; R₃ is C₁ -C₃ alkyl; each m is1 to 2; and n is 1 to 20;provided that at least one of R₂ is apolyethylene glycol substituent.
 70. A compound of claim 69 wherein eachR₂ is independently a polyethylene glycol substituent.